Sex and disease regulate major histocompatibility complex class I expression in human lung epithelial cells

Abstract Major histocompatibility complex class I (MHC I) molecules present peptides to CD8+ T‐cells for immunosurveillance of infection and cancer. Recent studies indicate lineage‐specific heterogeneity in MHC I expression. While respiratory diseases rank among the leading causes of mortality, studies in mice have shown that lung epithelial cells (LECs) express the lowest levels of MHC I in the lung. This study aims to answer three questions: (i) Do human LECs express low levels of MHC I? (ii) Is LEC MHC I expression modulated in chronic respiratory diseases? (iii) Which factors regulate MHC I levels in human LECs? We analyzed human LECs from parenchymal explants using single‐cell RNA sequencing and immunostaining. We confirmed low constitutive MHC I expression in human LECs, with significant upregulation in chronic respiratory diseases. We observed a sexual dimorphism, with males having higher MHC I levels under steady‐state conditions, likely due to differential redox balance. Our study unveils the complex interplay between MHC I expression, sex, and respiratory disease. Since MHC I upregulation contributes to the development of immunopathologies in other models, we propose that it may have a similar impact on chronic lung disease.


| INTRODUCTION
By scanning peptides presented by MHC I molecules, CD8+ T-cells perform immunosurveillance against infection and neoplastic transformation (Granados et al., 2015).MHC I expression also contributes to critical homeostatic functions within tissues, such as neuronal survival (Mangani et al., 2023).The cellular machinery responsible for MHC I surface expression is complex, involving peptide generation by proteasomes, followed by translocation into the endoplasmic reticulum through Transporter associated with Antigen Processing 1 and 2 (TAP1 and 2) (Blees et al., 2017).Assisted by various chaperone proteins, peptides are loaded onto MHC I complexes, consisting of an MHC I heavy chain and beta-2-microglobulin (B2M).Once formed, the MHC-peptide complex is transported to the cell surface via the Golgi apparatus, ready for interaction with immune cells.MHC I expression is transcriptionally regulated at several levels (Jongsma et al., 2019).NOD-like receptor family caspase activation and recruitment domain containing 5 (NLRC5) regulates basal and induced expressions of MHC I components.The MHC I promoter also has binding sites for NF-κB and IFN sensitive response elements, allowing regulation by NF-κB signaling and IFN regulatory factors (IRF), respectively.MHC I is ubiquitously present in nearly all nucleated cells.However, recent studies have revealed heterogeneity in MHC I expression across tissues and cell types (Boegel et al., 2018).Thus, quiescent tissue stem cells evade immune recognition by downregulating MHC I expression (Agudo et al., 2018).On the other hand, MHC I overexpression is instrumental in various immunopathologies, including neurodegenerative diseases, myopathies, type I diabetes, and inflammatory bowel diseases (Bär et al., 2013;Fréret et al., 2013;Hamilton-Williams et al., 2003;Zalocusky et al., 2021).Hence, MHC I expression must be tightly regulated because of its pleiotropic effects.
Lung diseases, as reported annually by the World Health Organization, are among the leading causes of death worldwide.Chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), and cystic fibrosis (CF) are prominent examples of chronic diseases characterized by disrupted lung epithelium function, susceptibility to infections, and heightened inflammation (Ruffin et al., 2018;Serezani et al., 2022;Villaseñor-Altamirano et al., 2023).COPD and IPF are progressive and principally affect older adults with a smoking history.In contrast, CF is a genetic disease caused by mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR) channel (Trinh et al., 2012).These diseases are heterogeneous and exhibit sex dimorphisms in severity and pathogenesis (Holtrop et al., 2021;McGee et al., 2014;Tam et al., 2016).We reported that constitutive MHC expression is very low in mouse lung epithelial cells (LECs) but can be upregulated by inflammation driven by inhalation of LPS (Benhammadi et al., 2020;Mathé et al., 2022).
We hypothesized that, as in mice, MHC I might be expressed at a low level in human LECs and be upregulated in chronic lung diseases.To address this hypothesis, we conducted comprehensive analyses using single-cell RNA sequencing (scRNA-seq) profiles obtained from lung parenchymal tissue of patients with COPD, IPF, CF, and non-diseased control donor lungs.We found a conspicuously low expression of MHC I in human LECs compared to other cell types in non-diseased lungs.LEC MHC I expression was significantly increased in COPD, IPF, and CF.Additionally, we unveiled a significant sex dimorphism in the expression of MHC I in healthy and diseased lungs.Modulation of MHC I expression could significantly impact the development of chronic lung diseases.

| Publicly available scRNA-seq data analyses
We analyzed 312,928 cells from 26 non-diseased, 18 COPD, and 28 IPF distal lung parenchyma explants publicly available (Adams et al., 2020).Sample processing and digestion procedures were described in the original paper.For CF donors, a Seurat object containing cells from 19 CF patients and 19 non-diseased control donors was downloaded (Carraro et al., 2021).The sex, age, and smoking status of the donors were obtained from the original papers and are presented in Table S1 (non-disease, COPD, and IPF donors) and Table S2 (CF patients).Additionally, scRNA-seq data from intestinal epithelium were examined (Smillie et al., 2019).All data were analyzed using Seurat V.4 (Butler et al., 2018).Quality control and cell annotation procedures followed the protocols outlined in the original studies (Adams et al., 2020;Carraro et al., 2021;Smillie et al., 2019).

GSEA analysis
Differential gene expression analysis was conducted using the Seurat function "FindMarkers", comparing each parameter (sex or disease) one by one for each cell type of interest.We used a natural log fold-change threshold of 0.25, a minimum percentage of cells expressing a gene of 10%, and the Wilcoxon Rank Sum test.DEGs obtained for each comparison are shown in Table S3.Gene Set Enrichment Analysis (GSEA) was performed using the "fgsea" function with Gene Ontology (GO) biological pathways terms as a gene set database from gsea-msigdb.org (Korotkevich et al., 2021).We used the default parameters.Additionally, GO term enrichment analysis from DEGs (Figure 6) was conducted using https:// www.panth erdb.org, using the GO biological process complete as a statistical overrepresentation test.

| Statistical analysis
For scRNA-seq data, statistical significance was evaluated with a non-parametric Wilcoxon rank sum test using the Seurat function "find markers".The type of comparisons is shown in the figure legends, and exact p values are indicated on each figure.Statistical significance was evaluated with an unpaired t-test using GraphPad Prism version 8 for immunofluorescence staining.

| Human LECs express constitutively low amounts of MHC I transcripts
To investigate MHC I expression in non-diseased human LECs, we analyzed scRNA-seq data from distal lung parenchyma from 21 healthy non-smoking donors (Adams et al., 2020).We quantified the expression of the three classical human MHC I alleles (HLA-A, HLA-B, and HLA-C) and B2M across four major lung cell types: epithelial, lymphoid, myeloid, and fibroblastic.We selected cMonocytes as a reference because they are professional antigen-presenting cells (APCs).Relative to cMonocytes, MHC I expression was superior in other lympho-myeloid cells and inferior in fibroblasts and LECs (Figure 1a).LECs from the bronchioles (ciliated, club, basal, and goblet cells) and alveoli (alveolar type I (ATI), and alveolar type II (ATII)) expressed less MHC I than all other lung cell populations (Figure 1a).Next, we compared the expression of genes crucial for MHC I surface expression and their transcriptional regulation (Figure 1b-e).Key players in the peptide loading of MHC class I, such as TAP1, Calnexin (CANX), Calreticulin (CALR), and TAP binding protein (TAPBP) were expressed at low levels in ECs (Figure 1b).LECs also exhibit low amounts of constitutive (PSMB5, PSMB6, PSMB7) and immunoproteasome (PSMB8, PSMB9, PSMB10) subunits (Figure 1c) and diminished IFNstimulated genes (ISGs) expression, including NLRC5, STAT1, STAT2, IRF1, JAK1, and JAK2 (Figure 1d).While most of these genes are expressed at low levels in LECs, the differences reached statistical significance only relative to lymphoid cells (Figure 1b-d).However, LECs express significantly higher levels of genes encoding NF-κB inhibitors (NFKBIA and NFKBIZ) and lower levels of NF-κB subunits coding transcripts (REL, RELB, and NFKB1) (Figure 1e).Together, these results suggest that low constitutive MHC I may be explained by repression of NF-κB signaling.
To pinpoint the key biological pathways that distinguish LECs from other lung cells, we conducted GSEA with DEGs obtained from the comparison between LECs and fibroblastic cells (Figure 1f), myeloid cells (Figure 1g), and lymphoid cells (Figure 1h).These analyses revealed a lower enrichment of the biological pathways: "activation of immune response" and "antigen processing and presentation of peptide antigen", in LECs compared to the three other cell types (Figure 1f-h).Genes included in these pathways are described in Table S5.These results reinforce that the low expression of genes linked to MHC I expression is a notable feature of LECs.

| Male LECs constitutively express more MHC I transcripts than females
Sex is one of the most significant sources of biological variability.Here, "sex" encompasses all biological differences between subjects genetically assigned as males and females.We conducted a sex comparative analysis of LECs obtained from non-diseased donors analyzed in Figure 1.
Males exhibited significantly more MHC I transcripts than females within the Ciliated, Goblet, and ATII cell subsets (Figure 2a).This observation was validated at the protein level through IF staining of cytopsin slides of primary airway and alveolar epithelial cells (Figure 2b,c), revealing significantly higher MHC I fluorescence intensity in ATII (pro-SPC+ staining) and secretory cells (which include club and goblet cells, MUC5AC+ staining) from males compared to females.No significant difference was observed in ciliated cells at the protein level, probably because of the lower number of this cell population stained on each slide (data not shown).Sex differences in MHC I expression were not found in non-EC lung cells (Figure S1a) but were observed in certain types of ECs from the gut (Figure S1b).Hence, (i) in the lung, only LECs show higher MHC I expression in males, and (ii) this epithelial cell sex dimorphism is not limited to the lung.
We then compared the expression of genes involved in MHC I processing in ciliated and ATII cells (LECs showing the most significant sex dimorphism at the transcriptomic level) from males and females.Males expressed significantly more peptide-loading complex (PLC) genes (CALR and CANX in ciliated cells and TAPBP in ATII) than females (Figure 3a).Interestingly, the immunoproteasome subunit PSMB9 expression was higher in males, while the constitutive subunit PSMB5 expression was higher in females (Figure 3b).Although NLRC5 and STAT1 tended to be expressed at higher levels in males and JAK1 in females, these differences were insignificant (Figure 3c).Moreover, males expressed more IFNGR, with significant differences observed for IFNGR2 in ciliated cells (Figure 3d).Of particular interest, molecules involved in the NF-κB pathway exhibited significant sex dimorphisms in their transcript levels.In ATII cells, NF-κB inhibitors were significantly more expressed in females than males (NFKBIA and NFKBIZ), whereas males generally expressed more NF-κB subunits in ATII and ciliated cells than females, which is significant in ciliated cells (REL and NFKB1) (Figure 3e).GSEA analysis revealed that males exhibited enrichment in biological pathways related to the immune response in ATII and ciliated cells.In contrast, female ciliated cells showed enrichment for pathways associated with their functional specificities (e.g., cilium movement and organization, which included HYDIN, DNAH5, DNAH7, and DNAH9) (Figure 3f).Regulation of reactive oxygen species (ROS) metabolism, which regulates NF-κB signaling and MHC I expression (Charni et al., 2010;Choi et al., 2021;Schmidt et al., 1995;Wang et al., 2016), ranked among the top five enriched pathways in male ATII cells (Figure 3f).The "Positive regulation of ROS metabolism" pathway was significantly enriched in male ATII and ciliated cells from males (Figure 3g).These results suggest that the sexual dimorphism in LEC MHC I expression is driven by differences in ROS production and, consequently, activation of the NF-κB pathway.

| Male LECs express higher amounts of genes involved in T-cell priming and cell death than females
In addition to MHC I presentation, priming of naïve CD8+ T-cells depends on the presence of costimulatory molecules on APCs (e.g., the more classical CD80 and CD86, as well as ICAM1, ICOS-L, and CD58) (Corbière et al., 2011).Without costimulatory molecules, cells expressing MHC I can be recognized by primed effector CD8+ T-cells but cannot prime naïve CD8+ T-cells.The efficiency of CD8+ T-cell responses is further amplified by concurrent stimulation of CD4+ T-cells by peptides presented by MHC II molecules (e.g., HLA-DR).Among LECs, ATII are recognized as APCs and express APC-specific markers, including HLA-DR (Gereke et al., 2009).A similar role has been proposed for ciliated cells (Rossi et al., 1990).We examined the expression of genes coding for costimulatory molecules in ATII and ciliated cells from males and females.Males expressed significantly higher levels of HLA-DR and CD86 than females (Figure 4a,b).On the other hand, females expressed more ICAM1 (usually found in cells lacking CD86) (Figure 4b).We examined T-cell phenotypes to  S4. Images were taken using a 40× objective.Scale bars, 30 μm.All donors (males and females) were stained and acquired simultaneously in the same conditions for each marker.No changes in the intensity or contrast were made.(c) Corrected total cell fluorescence (CTCF) calculated using the formula: integrated density-(area of selected cell × Mean fluorescence of background readings).The statistical significance of CTCF differences between males and females was determined using an unpaired t-test.Graphs show the means of all cells acquired in each group ± SD. assess whether disparities in MHC I and costimulatory molecules may influence T-cell responses.Male Tcells showed higher late activation markers (HLA-DR) expression than female T-cells (Figure 4c).Furthermore, GSEA analysis performed in ATII and ciliated cells confirmed the enrichment of genes involved in "T-cell activation" in males compared to females (Figure 4d and Table S5).Additionally, we observed an enrichment of genes related to the positive regulation of cell death in male ATII and ciliated cells compared to females (Figure 4e and Table S5).Hence, alongside higher MHC I expression, ATII and ciliated cells from males overexpress genes involved in T-cell priming and cell death.

| LECs increase MHC I expression in chronic respiratory diseases
In mice, inflammation triggered by LPS upregulates MHC I in LECs (Mathé et al., 2022).We, therefore, wished to evaluate whether this would also be the case for LECs in patients with COPD and IPF.COPD is characterized by a robust inflammatory response involving CD8+ T-cells (Maeno et al., 2007;Villaseñor-Altamirano et al., 2023).Conversely, IPF is marked by irreversible scarring of the distal lung, inflammation described as mild, and CD8+ T-cells correlate with the severity of the disease (Moore et al., 2014;Serezani et al., 2022).We initially compared the expression of the three MHC I alleles and B2M in LECs from COPD and IPF patients to that in non-diseased control donors.Each donor's age and smoking status are detailed in Table S1.We found a significant upregulation of MHC I expression in most LEC subsets from male and female COPD patients (Figure 5a,b).In IPF donors, the upregulation of MHC I transcripts was more restricted, particularly in males (panel a), where a significant increase in MHC I expression was observed only in Basal, Club, and ATI/II cells (Figure 5a,b).The increase in MHC I in COPD and IPF was less consistent among nonepithelial lung cell subsets.Among non-LECs, only lung fibroblasts showed a consistent rise in HLA-A, -B, and -C in males and females (Figure S2).Some leukocytes even exhibited a decrease in MHC I expression (Mature DCs in males with IPF; NK and T-cells in females with COPD or IPF).We conclude that the upregulation of MHC I in COPD and IPF is limited to non-hematolymphoid cells and is more conspicuous in LECs than fibroblasts.
We then looked at the expression of genes regulating the surface expression of MHC I (PLC and proteasome subunits) (Figure 5c,d) or its transcriptional regulation (IFN and NF-κB signaling) (Figure 5e-g).In COPD and IPF, male and female LECs showed no significant increase in PLC gene expression, except for TAPBP in females (Figure 5c).Expression of immunoproteasome subunits was increased in both sexes, with significant differences observed only in males with COPD (PSMB8 and 9) and females with IPF (PSMB9) (Figure 5d).NLRC5 expression was significantly higher in males and females with COPD, while other ISGs like STAT1 and IRF1 showed non-significant increases (Figure 5e).IPF patients exhibited no significant increase in ISG expression in either sex.Both males and females overexpressed IFNGR in COPD and IPF patients, but statistical significance was reached only in females for both diseases (Figure 5f).Additionally, both sexes exhibited increased expression of some NF-κB subunits (REL, RELB, or NFKB1) and decreased expression of NF-κB inhibitors (NFKBIA or NFKBIZ), indicating NF-κB signaling activation in LECs in both pathologies (Figure 5g).
COPD and IPF are acquired diseases, prompting us to investigate whether a similar increase in MHC I expression occurs in genetic respiratory diseases like CF.We analyzed scRNA-seq data from CF distal lungs (bronchi and bronchioles), which were publicly available (Carraro et al., 2021).As sex information was unavailable for all individuals (Table S2), we merged male and female patients.Our analysis revealed increased HLA-B expression in ciliated, club, and goblet cells from patients with CF (Figure S3).We speculate that the rise in HLA-A and C expression might be attenuated due to the merging of males and females exhibiting sex dimorphisms in this analysis.Using IF staining of primary human alveolar and airway epithelial cells, we confirmed the increase in MHC I expression at the protein level in ATII and secretory cells from CF patients (Figure S4 and Table S4).
Our findings demonstrate increased MHC I expression in LECs across three chronic respiratory diseases: COPD, IPF, and CF.This increase is likely due, at least in part, to IFN stimulation and NF-κB signaling in COPD and IPF.

| MHC I expression is differentially regulated in males and females with COPD and IPF
LECs showed higher constitutive MHC I levels in males than females (Figure 2).We therefore wished to investigate whether this difference persisted in the context of chronic respiratory diseases like COPD and IPF.We observed a significantly higher MHC I expression in males than females across all LEC subsets in the COPD donor group (Figure 6a).In IPF, the differences between sexes affected ATII and club cells and showed a different directionality (higher expression in females) (Figure 6b).Hence, males present a higher MHC I increase in COPD, whereas upregulation of MHC I in IPF is more marked in females (Figure S5a,b).
To understand the differential regulation of MHC I between males and females in COPD and IPF, we analyzed DEGs from ATII cells.We focused on ATII cells since the sex differences in this cell type were most consistent and striking throughout our study.Biological pathway analysis revealed that in COPD patients, male ATII cells exhibited enrichment in pathways related to antigen presentation and ROS generation (e.g., superoxide anion generation, mitochondrial respiration, Figure 6c and S5C-F).Conversely, female ATII cells showed enrichment in their specific function-related pathways (e.g., surfactant

F I G U R E 5
LECs upregulate MHC I-coding transcripts in chronic respiratory diseases.Expression levels of MHC I alleles and B2M transcripts in COPD (green), IPF (red), and non-diseased control donors (gray) in males (a) and females (b).The control group comprises 12 females and 14 males, the COPD group contains 9 females and 9 males, and the IPF group has 6 females and 22 males.All the groups included smokers and non-smokers.Average expression and percentage of LECs expressing genes involved in the PLC (c), proteasome subunits (d), ISGs involved in the transcriptomic regulation of MHC I (e), IFN receptors (f), and NF-κB signaling (g).All left panels display male data, and all right panels show female data.Statistical significance was assessed using a non-parametric Wilcoxon test comparing the average expression of each transcript in LECs from COPD or IPF patients versus non-diseased control donors.LECs analyzed in panels c-g contained merged LEC subtypes (ATI, ATII, basal, club, ciliated, and goblet cells), as in Figure 1.
F I G U R E 6 MHC I expression is differentially regulated in males versus females with COPD and IPF.Expression levels of MHC I alleles and B2M in males (darker color) and females (lighter color) in COPD (n = 9 males and 9 females) (a) and IPF (n = 22 males and 6 females) (b).Statistical significance was assessed using a non-parametric Wilcoxon test comparing the average expression of each transcript in males versus females.GO biological pathway enriched in ATII cells from males (c) and females (d) with COPD and males (e) and females (f) with IPF.GO terms were selected according to their fold enrichment (superior to 10 for "COPD Male ATII" and "IPF Female ATII" and superior to 7 for "COPD Female ATII" and "IPF Male ATII").
homeostasis, including SFTPD and ABCA3 genes, and regulation of hippo signaling, including YAP1 and SAV1 genes) (Figure 6d).In IPF, male ATII cells showed enrichment of genes involved in surfactant homeostasis and regulation of hippo signaling pathways (Figure 6e), and female ATII in pathways related to MHC assembly and mitochondrial respiration (Figure 6f and S5c-f).DEGs associated with ROS generation and mitochondrial respiration correlated with HLA-B expression in males and females, emphasizing their putative role in regulating MHC I expression (Figure S5c-f).These findings show that inherent differences in ROS levels between males and females correlate with MHC I expression in LECs at steady state and in chronic respiratory diseases.To assess the potential impact of these disparities on T-cell activation in COPD and IPF, we examined genes involved in T-cell priming in ATII and ciliated cells.In COPD, male ATII cells exhibited elevated levels of HLA-DR, CD86, and CD58 (Figure 7a,b).Conversely, in IPF, females displayed increased HLA-DR and CD86 levels, with males exhibiting higher ICAM1 expression (Figure 7a,b).Furthermore, male T-cells exhibited significantly higher levels of the late activation marker HLA-DR.Besides, females with COPD showed elevated levels of the early activation marker CD69 (Figure 7c).IPF donors showed no significant differences in T-cell activation markers (Figure 7c).Finally, GSEA performed in ATII from males and females showed T-cell activation (Figure 7d) and cell death-related gene (Figure 7e) enrichment in males with COPD and females with IPF (Table S5).
These findings underscore significant sex-based differences in the expression of genes related to MHC I, costimulatory molecules, and cell death markers among non-diseased individuals and those with COPD and IPF, with variations specific to each disease.These variations also extend to genes coding for T-cell activation markers.

| DISCUSSION
The lung faces constant exposure to airborne pathogens and particles, rendering it susceptible to inflammationinduced damage.Therefore, the lung must maintain a delicate balance: rapid clearance of pathogens without undue harm to LECs.Accordingly, dysregulated tissueimmune interactions are the hallmark of diverse chronic lung diseases (Singanayagam et al., 2019;Vijayakumar et al., 2022;Wang et al., 2023).This study delves into the factors governing MHC I expression and the linked susceptibility to CD8+ T-cell mediated damage in healthy and diseased LECs.
MHC I expression is transcriptionally regulated by ISGs (particularly NLRC5) and NF-κB (Jongsma et al., 2019).The basal expression of MHC I is affected primarily by ISGs in some cell types (e.g., neurons) and by NF-κB in other cell types (e.g., astrocytes and oligodendrocytes) (Massa et al., 1993).We report that, like mouse LECs (Mathé et al., 2022), human LECs constitutively express very low levels of MHC I. Also, human LECs express low amounts of NLRC5, other ISGs, and various genes responsible for MHC processing.However, compared to other lung cell types, MHC I expression in LECs correlated more strongly with decreased NF-κBrelated gene expression.
Notably, we found that in chronic lung diseases, MHC I upregulation was strong in LECs, mild in fibroblasts, and absent in lympho-myeloid cells.Thus, chronic lung inflammation modulated MHC I expression primarily in the cell type with the lowest basal expression (LECs).This is reminiscent of other studies where disease correlated with MHC I upregulation in cell types with low basal expression, such as neurons, myocytes, and enterocytes (Bär et al., 2013;Fréret et al., 2013;Zalocusky et al., 2021).This suggests that fluctuations in MHC I levels in chronic inflammatory settings predominantly affect cells with low basal expression.Global MHC upregulation increases the abundance and diversity of MHC-associated peptides and could pave the way to autoimmunity (Caron et al., 2011;Stern et al., 2024).Coherent with this idea, the lungs of patients with COPD and IPF commonly exhibit increased CD8+ T-cells (Maeno et al., 2007;Papiris et al., 2007;Serezani et al., 2022;Villaseñor-Altamirano et al., 2023).Mechanistically, our transcriptomic analyses of LECs revealed an activation of NF-κB signaling in COPD and IPF.Upregulation of NLRC5 and IFNGR was limited to COPD, where inflammation is more intense than in IPF.
LPS-induced MHC I upregulation in mice correlates with the downregulation of genes crucial for LEC functions, such as cell differentiation (ATII) and cilium movement (ciliated cells) (Mathé et al., 2022).Likewise, in human LECs, we observed an enrichment of genes implicated in epithelial cell functions in conditions where MHC I expression was low.ATII from females in COPD and males in IPF are enriched in genes involved in hippo signaling (YAP1, crucial for their differentiation in ATI) (LaCanna et al., 2019) and surfactant homeostasis (SFTPD and ABCA3).In contrast, ciliated from healthy females are enriched in genes involved in cilium movement (HYDIN, DNAH5, DNAH7, and DNAH9).Thus, overexpression of MHC I may be a marker of defective epithelial cell functionality.The mechanistic underpinnings of this correlation have yet to be elucidated.
An unexpected finding from our study is the higher expression of MHC I observed in male ATII, goblet, and ciliated cells compared to their female counterparts.The superior MHC I expression in male LECs is accompanied by the upregulation of genes involved in T-cell priming.In non-diseased LECs, this sex dimorphism aligns with elevated levels of NF-κB subunits in males and NF-κB inhibitors in females.Various stimuli, such as cytokines, hormones, and cellular stress, can modulate NF-κB signaling (Bartlett et al., 2012;Capece et al., 2022).Cellular stress, specifically ROS production, emerges as one of the top five enriched biological pathways in male ATII and ciliated cells.The role of ROS in regulating MHC I expression has been extensively studied in DCs and tumor cells (Charni et al., 2010;Choi et al., 2021;Wang et al., 2016).Considering the enrichment of genes related to ROS production in male LECs and the known role of ROS in regulating MHC I expression, we posit that sex differences in the redox balance of the lung contribute to the observed sex-specific regulation of MHC I expression in healthy LECs.ROS have been found to elevate the expression of PLC and immunoproteasome subunits (Liu et al., 2017).In accordance with this, we found that these genes are expressed at higher levels in male LECs than in females.Sex hormones play pleiotropic roles in physiological differences between males and females.Notably, estrogens have been shown to inhibit NF-κB signaling (Xing et al., 2012), the master regulator of MHC I expression in human LECs.Additionally, estrogens suppress ROS production by modulating antioxidant enzyme activity, whereas testosterone exhibits pro-oxidant properties (Celestino et al., 2018;Chignalia et al., 2015;Malorni et al., 2008;Reed & Arany, 2014).As estrogen receptors' expression in the lung epithelium is well-documented (Mollerup et al., 2002), we propose that sex hormones contribute to the sex-specific regulation of MHC I in LECs by modulating ROS production and NF-κB signaling pathways.
To our knowledge, sex dimorphism in the expression of MHC I-related genes has been reported in two contexts: the aging brain, where aging females express higher levels of MHC I than males, and in peripheral blood mononuclear cells, where immunoproteasome activity is lower in females compared to males (Kammerl et al., 2021;Mangold et al., 2017).These two studies raised the possibility that dimorphisms of MHC I expression play a role in the differences between males and females in pathological contexts.Also, they indicate that inter-sex differences in MHC I regulation may not have the same directionality in all cell types.Chronic respiratory diseases exhibit notable sex differences in severity and pathophysiology (Hardin et al., 2017;Holtrop et al., 2021;McGee et al., 2014).We documented that, in COPD, males show a more pronounced upregulation of MHC I and antigen presentation-related genes than females.The opposite trend is observed in IPF.Interestingly, in both diseases, the sex exhibiting higher MHC I expression shows enrichment in genes associated with mitochondrial respiration and ROS production.The involvement of redox balance in sex dimorphisms related to diseases has been demonstrated across various contexts, including COPD (Celestino et al., 2018;Tam et al., 2016).Furthermore, oxidative stress plays a significant role in COPD, CF, and IPF (Murthy et al., 2009;Velsor et al., 2006;Yasuoka et al., 2019;Zuo et al., 2014).The most parsimonious model to explain these data is that sex hormones influence the sex-specific regulation of MHC I expression by the redox balance and contribute to the sex differences in chronic respiratory diseases.
We acknowledge that our study presents several limitations.First, it relies mainly on transcriptomic data.
The limited availability of human lung tissue samples has posed challenges for conducting further protein analysis in patients with COPD and IPF.Significant age differences exist between the control and COPD/IPF groups, reflecting the typical elderly population affected by these conditions (female median age: 48 for control, 60 for COPD, and 66.5 for IPF; male median age: 43.25 for control, 65 for COPD, and 66.5 for IPF).We chose not to exclude younger donors from the control group to avoid compromising the sample size.Other patient characteristics may be associated with worse outcomes in these pathologies, although the mechanisms are unclear (Maselli et al., 2019).This study focused on specific criteria such as sex and disease type.Future studies should consider patient characteristics and investigate differences in MHC I expression based on clinical epidemiology (age, comorbidity, asthma/COPD overlap, smoking history, chronic bronchitis, or exacerbation).Finally, the functional implications of MHC I overexpression on lung epithelium have yet to be worked out.Future investigations, encompassing murine models and human subjects, should strive to elucidate the active role of MHC I in LEC biology, which we believe could be crucial to their function.For example, ROS activate cell senescence (Salminen et al., 2012), which plays a role in COPD and IPF (Aoshiba & Nagai, 2009;Barnes et al., 2019;Hara et al., 2012).Cell senescence is an irreversible state of cell cycle arrest that is typically physiological and protective but can contribute to pathological mechanisms when excessive.Previous work has highlighted the immunogenic role of senescent cells, particularly in the activation of interferon signaling, CD8+ T-cells, and MHC I expression (Marin et al., 2023).Thus, cell senescence may contribute to MHC I elevation in COPD and IPF patients and should be considered in future studies investigating MHC I.
In summary, our study offers novel insights into the regulation of MHC I in both healthy and diseased human lungs.It illustrates how MHC I expression and the immune context, in general, are modulated by multiple factors that vary from one cell type to another in a sexspecific manner.Furthermore, our work raises several questions warranting further investigation.Two may be a priority.First, does MHC I upregulation in LECs lead to immune-mediated damage in chronic inflammatory lung diseases?Second, does the low basal MHC I expression in LECs hamper cancer immunosurveillance?AUTHOR CONTRIBUTIONS JM, SB, and CP conceptualized the study and drafted the initial manuscrpt.JM conducted the experiments and performed the data analysis.JM and SB discussed the findings and prepared the figures.EB and DA assisted in designing, analyzing, and discussing the immunofluorescence analysis and provided the human cytospin slides from healthy subjects and cystic fibrosis patients.CP, SB, and EB supervised the study.All authors reviewed the manuscript.

F
I G U R E 1 Human LECs constitutively express low amounts of MHC I transcripts.(a) Comparison of MHC I alleles (HLA-A, HLA-B, HLA-C) and B2M transcript expression in scRNA-seq data from 21 healthy non-smoking donors, including 11 females and 10 males.The expression level corresponds to the feature counts for each cell divided by the total counts for that cell and multiplied by the scale factor (1000), then natural logtransformed using log1p.Cell types are ordered from the highest MHC I expression to the lowest.Assessment of the average expression and the percentage of cells expressing PLC genes (b), constitutive and immunoproteasome subunits (c), ISGs involved in MHC I transcriptional regulation (d), and NF-κB signaling (transcription factors: REL, RELA, RELB, NFKB1; or inhibitors: NFKBIA, NFKBIZ, NFKBID) (e).Dot size reflects the percentage of cells with gene expression; color corresponds to the degree of expression.Statistical significance was assessed using a non-parametric Wilcoxon test comparing LECs and cMonocytes (a) or the average expression of each transcript in LECs relative to each other cell type (lymphoid, myeloid, or fibroblastic cells) (b-e).GSEA focusing on two GO biological pathways, "activation of immune response" (upper panels) and "antigen processing and presentation of peptide antigen" (lower panels) in LECs compared to fibroblastic cells (f), myeloid cells (g), and lymphoid cells (h).Normalized enrichment score (NES) and adjusted p-value (p adj) are shown on the graph when significant.

F
LECs express more MHC I in males than females in non-diseased lungs.(a) Male (n = 10) and female (n = 11) comparison of MHC I alleles and B2M mRNAs expression levels in the healthy non-smoking donors analyzed in Figure 1.Statistical significance was assessed using a non-parametric Wilcoxon test comparing male and female LECs.(b) IF staining of human ATII (Pro-SPC+) and secretory (MUC5AC+) cells with pan anti-HLA-ABC antibody.Representative images for all staining conditions of non-diseased LECs from males (n = 3) and females (n = 3).Donors' data are shown in Table

F
Male LECs express more transcripts contributing to MHC I expression than females.Percentage and average expression of ciliated and ATII cells expressing genes involved in the PLC (a), proteasome subunits (b), ISGs involved in MHC I transcriptional regulation (c), IFN receptors (d), and NF-κB signaling (e).Statistical significance was assessed using a non-parametric Wilcoxon test comparing the average expression of each transcript in male (n = 10) versus female (n = 11) healthy non-smoking donors for each cell type (ATII and ciliated cells).(f) GSEA showing the top five enriched GO biological pathways in ATII and ciliated cells from males (blue) and females (purple).(g) Enrichment score of the GO biological pathway "Positive regulation of reactive oxygen species metabolism" in male versus female ATII and ciliated cells.NES and p adj are shown in the figure.

F
Male LECs express higher amounts of transcripts coding for T-cell activation and cell death markers than females.Percentage and average expression of ciliated and ATII cells expressing genes coding for HLA-DR (a) and costimulatory markers (b).(c) Percentage and average expression of T-cell activation markers.Statistical significance was assessed using a non-parametric Wilcoxon test comparing the average expression of each transcript in male (n = 10) versus female (n = 11) healthy non-smoking donors for each cell type (ATII, ciliated, and T-cell).Enrichment score of the GO biological pathways "T-cell activation" (d) and "positive regulation of cell death" (e) in ATII and ciliated cells from males and females.NES and p adj are shown in the figures.

F
I G U R E 7 LECs differentially regulate transcripts coding for T-cell activation and cell death markers in males versus females with COPD and IPF.Average expression and percentage of ATII and ciliated cells expressing HLA-DR (a) and costimulatory molecules (b) in males and females with COPD (n = 9 males and 9 females) and IPF (n = 22 males and 6 females).(c) Average expression and percentage of T-cell expressing T-cell activation markers with COPD and IPF.Statistical significance was assessed using a non-parametric Wilcoxon test comparing the average expression of each transcript between males and females in each cell type (ATII, ciliated, T-cell).Enrichment score of the GO biological pathways "T-cell activation" (d) and "positive regulation of cell death" (e) in ATII cells from male compared to female patients with COPD or IPF.NES and padj are shown in the figures.